B-cell non-Hodgkin lymphomas (NHL) are the sixth most common cause of cancer-related deaths in the United States. While many patients with aggressive lymphomas may be cured with cytotoxic therapy, most indolent lymphomas are incurable with current therapy. Novel effective therapies are therefore needed to treat these patients. The goal of our research is to develop novel biologic therapies for patients with B-cell NHL by utilizing strategies that augment the immune response to the malignant B-cell. During the previously funded period we evaluated whether adding IL-12, an immunostimulatory cytokine, to rituximab in patients with B-cell lymphoma would increase the antibody dependent cytotoxicity of rituximab. In the clinical trial, we found that IL-12 did not significantly increase the response rate above what would be expected with rituximab alone. In correlative studies we found that IL-12 resulted in upregulation of genes in peripheral blood cells but did not have similar effects in the tumor. In laboratory studies, we found that the lack of effector cell response to immune stimulation with IL-12 was related in part to the presence of intratumoral T-cells with suppressive function. Recent studies have suggested that CD4+CD25+ regulatory T (Treg) cells are involved in the regulation of anti-tumor immunity by inducing peripheral tolerance to tumor specific antigens. However, there are little data regarding the effect of Treg cells on tumor-specific T cell immunity in B-cell NHL and subsequently on the malignant B-cell growth. In preliminary studies, we have identified a subset of CD4+CD25+ T cells with a Treg cell phenotype that are present in B-cell NHL. In addition, we find that these Treg cells have the ability to suppress tumor-infiltrating CD4+ and CD8+ T cells in B-cell NHL and that they migrate in response to chemokines such as CCL22 produced by the malignant B-cells. Our central hypothesis is that tumor Treg cells contribute to the growth of malignant lymphoma B cells by suppressing tumor-infiltrating T cells and that malignant B-cells play an active role by selectively recruiting Treg cells to the areas of B-cell NHL. We therefore propose to firstly determine the mechanism by which these Treg cells are recruited to the malignant B-cell microenvironment in non-Hodgkin lymphoma and to discover whether they gain suppressive activity when present in the tumor microenvironment (Aim 1). Secondly, we will assess whether malignant B-cells interact directly with Treg cells in the tumor microenvironment and thereby orchestrate tolerance to their presence (Aim 2). Thirdly, we will establish whether depletion of intratumoral Treg cells, and inhibition of malignant B-cells to decrease Treg cell recruitment, will result in clinical benefit for patients with B-cell NHL (Aim 3). We anticipate that the proposed research will provide a better understanding of the Treg cell-mediated effects in B-cell malignancies. We also anticipate that the clinical use of denileukin diftitox, an interleukin-2 and diphtheria toxin fusion protein, in combination with rituximab, an anti-CD20 monoclonal antibody, will inhibit Treg cells in B-cell lymphoma patients and will also deplete lymphoma B-cells in malignant lymph nodes thereby preventing further recruitment of Treg cells into areas of B-cell lymphoma. This treatment combination will lead to a novel therapeutic approach to modulating Treg cells that will result in clinical benefit for patients with B-cell NHL.